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Low oxygen concentration occurs in a wide range of aquatic systems and range in temporal frequency, seasonality and persistence. These have always been naturally occurring low oxygen habitat but anthropogenic activities related primarily to organic and nutrient enrichment have led to increase in hypoxia and anoxia both in fresh as well as marine system. Freshwater systems are more frequently faced with low oxygen condition and fishes in a tropical country like India are quite frequently exposed to this. The general public is aware of the results of hypoxia as the phenomenon of “Fish Kills” occurring frequently in natural waters.

Aquatic organisms which are frequently exposed to hypoxia show adaptations at behavioural, morphological and physiological levels. To assess the effect of hypoxia at physiological level, change in hematological and blood parameters in selected tissues of cat fish, Mystus seenghala and carp fish Cyprinus carpiowere undertaken. Fish were exposed to experimentally provoked hypoxia for different duration and were sacrificed to study the effect of hypoxia on selected blood parameters in heart, liver, brain and muscle. Significant changes were recorded. The observations indicate that different tissues respond differently to the stress of hypoxia and the blood parameters respond in a tissue specific manner.

This study aims to analyse the comparative responses of aerobic and anaerobic enzyme activity and protein profiling to different degrees of hypoxia with two respiration patterns in fishes, Mystus seenghala and Cyprinus carpio.

Effect of oxygen deficiency on fish had drawn the attention of scientists as early as 1920s and extensive literature is available on fish during that period. Story of studies of adaptations of fish to low oxygen was extended by investigation undertaken in swamps (Carter and Beadle, 1931). A comprehensive study has been made on a number of freshwater, estuarine and marine fishes by Davis (1975) to record the minimum oxygen requirements for survival and growth of fishes. Greaney et al., (1980); Taylor and Miller, (2001); Pichavant et al., (2003) studied the effects of chronic (weeks of) hypoxia on oxygen carrying capacity.

Bushnell et al., (1984) investigated the effect ofchronic hypoxia on fish swimming performance and metabolism. The effect of hypoxia on swimming activity of fishes was supported by Dahlberg et al., (1968), Bushnell et al., (1984).Weber & Kraemer (1983) described that feeding and growth (Cech et al., 1984; Bejda et al., 1992; Secor & Gunderson, 1998; Taylor & Miller, 2001) are reduced in fishes when exposed to chronic hypoxia (≤3.0 mg O₂l¹).

Dunn & Hochachka (1986) and Dalla Via et al. (1998) observed in their studies that a metabolic reorganization takes place as a result of hypoxia that tends to follow one of two generalized patterns: (i) either the rate of anaerobic ATP production increases (Pasteur effect) or (ii) the ATP rate declines (metabolic depression). Chabot and Dutil, (1999) and Pichavant et al., (2003)studied the effects of chronic (weeks of) hypoxia on food intake.Metabolic correlation and comparative study in various fishes with different respiratory patterns were performed (Kumar 2016;Kumar 2017; Kumar et al., 2020 and Kumar 2021¹; Kumar 2021²). 

Live specimens (6 fishes) of Cyprinus carpio and Mystus seenghala(80-90 g 20-24 cm), were procured from a local market and were acclimatized at normoxia (7.2±0.3 mg/L, DO), at least for a month in tanks of 100 L capacity filled with 25 L of water at 25±3°C. They were fed once a day with processed feed of goat liver or flesh and soybean powder. Feeding was stopped 48 h before the start of experiment.

All the fishes were held for 12 hrs duration of experimentally provoked hypoxia at three different levels:

(i)             65%-40%Oxygen saturation or 5.0±0.3 mg/l to 3.5±0.3 mg/l O₂ (Slight Hypoxia)

(ii)            40%-20% Oxygen saturation or 3.5±0.3 mg/l to 1.5±0.1 mg/l O₂ (Moderate Hypoxia) and

(iii)           Below 20%Oxygen air saturation or ≤1.5±0.1 mg/l O₂ (Severe Hypoxia)

Three separate experiments were carried out in the closed respirometer (without access to air). Decrease in dissolved oxygen (DO) was accomplished by bubbling nitrogen directly into the water of the experimental tank, or into the reservoir that supplied water to the respirometer. DO probe (WTW, CellOx 325) and pH meter (pH electrode; WTW, SenTix® 41-3) were installed to record dissolved oxygen (DO) and pH.

Hematologic Parameters

Fish were anaesthetized prior the collection of blood samples to reduce the handling stress during normoxia. Heparinized blood was used for erythrocyte counts, haemoglobin estimation and haematocrit (Hct) evaluation. Erythrocyte count was made with the help of Neubaur’s haemocytometer using standard diluents. Haemoglobin was estimated by the method of Blaxhall and Daisley (1973). [Hct] was determined following centrifugation of microhematocrit capillary tube filled with blood, at 10,000 rpm for 5 min (Assendelft and England 1982). Erythrocytic indices like mean corpuscular volume (MCV) mean corpuscular haemoglobin (MCH) Mean cell haemoglobin concentration (MCHC) was measured by Wells and Weber (1991).

In Mystus seenghala

TABLE-1: Haematological changes in Mystusseenghalaexposed to different level of hypoxia. Values are mean of three replicates±standard error of mean. 

 

FIGURE-1: Haematological parameters in blood of Mystusseenghalaexposed to varying oxygen concentration i.e. different hypoxia stages for 12 hours duration.(A) RBCs (10⁶×mm³), (B) Hb (gm/100 ml), (C) Hct (per deciliter), (D) MCV (fl/cell), (E) MCH (Pg/cell) and (F) MCHC (gm/decilitre).Asterisk (*) represents significant differences (p<0.05) between normoxia and different hypoxia stages.

In Cyprinuscarpio

TABLE-2: Haematological changes in Cyprinuscarpioexposed to different level of hypoxia. Values are mean of three replicates±standard error of mean.

                                                                               

                                                           

FIGURE-2: Haematological parameters in blood of Cyprinuscarpio exposed to varying oxygen concentration i.e. different hypoxia stages for 12 hours duration.(A) RBCs (10⁶×mm³), (B) Hb (gm/100 ml), (C) Hct (per deciliter), (D) MCV (fl/cell), (E) MCH (Pg/cell) and (F) MCHC (gm/decilitre).Asterisk (*) represents significant differences (p<0.05) between normoxia and different hypoxia stages.

In the present study on non-air-breathing catfish Mystus seenghala, an increase in [Hb] and [Hct] and decrease in MCHC in hypoxic conditions with mean values of [Hct] after moderate and severe exposure to hypoxia, suggested the possibility that oxygen carrying capacity of the blood might be enhanced by bringing more red blood cells into circulation. These cells are most likely released from the spleen upon adrenergic and/or cholinergic stimulation (Nilsson and Grove, 1974). These hormones serve to increase the transfer of oxygen across the gills and the transport of oxygen in the blood to actively metabolizing tissues.

During environmental hypoxia, catecholamines are mobilized into the blood when the arterial oxygen content significantly decreases (Perry and Reid, 1974). Evidence from teleost fish suggests that the release of red blood cells via splenic contraction does occur in response to elevated catecholamines (Nilsson et al.1975). Splenic contraction has been well characterized in fishes in response to hypoxia (Lai and Todd, 2006).

Extended holding of common carp, Cyprinuscarpio at low DO induced an improved ability to transport oxygen in blood relative to fish held at higher oxygen concentrations. Concentrations of both Hct and Hb were significantly higher in Cyprinuscarpioheld at low oxygen for 50 days relative to fish held at higher oxygen. Hct is the percentage of packed red blood cells relative to the whole volume of blood, but does not account for the size or number of erythrocytes. Hb is a quantification of the O₂ binding protein found in red cells, whereas MCHC is a measure of the Hb in a given volume of packed erythrocytes (Houston, 1990). Increases in Hct and/or Hb are typically caused by an increase in the production of erythrocytes, swelling of the erythrocytes, or a combination of both. These changes are typically a result of catecholamine releases that induce the release of erythrocytes from the spleen (Jensen et al. 1993), or acidosis in the blood, which alters the affinity of Hb to bind oxygen, and can stimulate an increase in erythrocytes (Wells, 2009). Increases in Hb and Hct concentrations between the air-breathing and non-air-breathing groups during an oxygen challenge may have been driven by the release of erythropoietin, the hormone responsible for synthesizing erythrocytes and releasing erythrocyte stores from the spleen. This is evidenced by the increase of erythrocytes numbers (i.e., increase in Hct and Hb) without increasing the amount of Hb per cell volume (i.e., no change in MCHC). This is only offered as a potential mechanism as erythropoietin was not quantified.

Rainbow trout (Oncorhynchus mykiss) subjected to sustained hypoxia (maximum 216 h) had persistent increases in erythropoietin, as well as increased Hb levels (Lai et al.2006), thereby providing an improved ability for oxygen uptake. Additionally, long-term exposure to hypoxia increases both Hb and Hct concentrations for numerous fish species, both air and water breathers (Scott and Rogers, 1981; Tun and Houston, 1986; Petersen and Petersen, 1990 and Timmerman and Chapman, 2004). These changes typically confer an increase in oxygen-binding affinity or increased substrata for oxygen binding on the erythrocyte, improving performance of fish in low oxygen conditions. Despite Hct and Hb concentrations not differing between control treatments for these two groups, Mystus seenghalaand Cyprinuscarpioacclimated to a low oxygen environment were able to increase those hematological variables relative to the high oxygen group following a low oxygen challenge(Kumar A. 2018; Kumar A. 2021; Kumar A. 2022).It is likely that this increase in Hb and Hct provided an increase in performance during hypoxia, but additional work measuring blood gas concentration and/or Hb/O2 affinity would be necessary to confirm this (Kumar A. & Gopesh A. 2015; Kumar A. 2016; Kumar A. 2017).

While these parameters, such as the description of regulation of blood physiological parameters, enzyme levels and their tissue expression, ventilation adjustments, adjustments of haematological parameters, ion regulation and behaviour have already been addressed by several authors, the relationship between these adaptive strategies and their occurrence among related fish groups is barely understood.Furthermore, no comparative account is available on hypoxia tolerance of fishes of different respiratory habits. The ability to compare these features not only among fishes but Vertebrates, including human is of paramount importance for the comprehensive understanding of breathing mechanisms under stress (Farrel and Rechards, 2009).

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